Lecture 6- Applied Enzyme Catalysis

Lecture 6-Applied Enzyme Catalysis Dr. A.K.M. Shafiqul Islam School of Bioprocess Engineering 15.01.2010

Inside the Chapter • Survey some of the applications of enzymes • Enzymatic hydrolysis and concept: Starch and Cellulose • Examine immobilized enzyme catalyst formulations • which allow sustained, continuous use of the enzyme.

Sources Enzymes • There are three major sources of enzyme • Plant • animal • or microbial • Although all living cells produce enzymes, one of the three sources may be favored for a given enzyme or utilization

Animal Sources • Some enzymes may be available only from animal sources. • Enzymes obtained from animals may be relatively expensive, e.g., rennin obtain from calf's stomach, • the value depend on demand of lamb or beef, • and their availability.

Plant Sources • While some plant enzymes are relatively easy to obtain e.g., papainfrom papaya • their supply is also governed by food demands

Microbial Enzymes • Microbial enzymes are produced by methods which can be scaled up easily. • Recombinant DNA technology now provides the means to produce many different enzymes, including those not normally synthesized by microorganisms or permanent cell lines, in bacteria, yeast and cultured cells.

Some Enzymes of Industrial Importance

Microbial Enzymes • Due to the rapid doubling time of microbes compared with plants or animals • microbial processes are attuned more easily to the current market demands for enzymes. • On the other hand, • for use in food or drug processes, only those microorganisms certified as safe may be exploited for enzyme production.

Microbial Enzymes • Although most of the enzymes used today are derived from living organisms, they are utilized in the absence of life • Example – extracellular enzymes, • secreted by cells in order to degrade polymeric nutrients into molecules small enough to permeate cell walls. • Grinding, mashing, lysing, or otherwise killing and splitting intracellular enzymes, • which are normally confined within individual cells.

Enzyme Kinetic • The enzyme kinetics study generally carried out with the purest possible enzyme preparations. Such research involves • the fewest possible number of substrates (one if achievable) • a controlled solution with known levels of activators (Ca2+, Mg2+,pH etc.), • cofactors, • and inhibitors.

Industrial enzymes • Many useful industrial enzyme preparations are not highly purified. • They contain a number of enzymes with different catalytic functions and are not used with or a completely defined synthetic medium. • Also, the simultaneous use of several different enzymes may be more efficient than sequential catalysis by a separated series of the enzymes. • such enzyme preparations are kinetically more simple than the integrated living organisms from which they are produced either a pure substrate

Hydrolytic Enzymes • Hydrolytic enzymes are normally associated with degradative reactions, e.g., • conversion of starch to sugar, • proteins to polypeptides and amino acids, • and lipids to their constituent glycerols, fatty acids and phosphate bases

Application of Hydrolytic Enzymes • In macroscopic degradations such as • food spoilage • starch thinning, • and waste treatment, • Also in the chemistry of • ripening picked green fruit • self-lysis of dead whole cells (autolysis), • desirable aging of meat, • curing cheeses, • preventing beer haze, • texturizing candies, • treating wounds, • and desizing textiles.

Hydrolytic Enzymes • The three groups of enzymes. Those involved in the hydrolysis of • ester, • glycosidic, • and various nitrogen bonds.

Hydrolytic Enzymes • Enzymes are named according to the chemical reactions they catalyze, rather than according to their structure.

Classification of Hydrolytic Enzymes

Application of Hydrolytic Enzymes • One-enzyme – one-reaction uniqueness does not generally exist, Enzymes from different plant or animal sources which catalyze a given reaction will not always have the same molecular structure or necessarily the same kinetics. • Consequently, • maximum reaction rate, • Michaelis constant, • pH of optimum stability or activity, • and other properties – depend on the particular enzyme source used.

Application of Hydrolytic Enzymes Many hydrolases are directed to specific compartments separated from the cytoplasm by membranes. This serves the purpose of protecting essential cytoplasmicbipolymers from degradation. Example, • Gram-positive bacteria secrete a variety of hydrolases into their environment. With their double membrane outer envelope, gram-negative bacteria have available the periplasmic space which safely stores a variety of hydrolases.

Application of Hydrolytic Enzymes • In eucaryotes, hydrolases may be stored inside the cell in membrane-enclosed lysosome organelles, reside in the periplasm in microbes like yeast, or be secreted into the environment. • Most hydrolytic enzymes used commercially are extracellular microbial products.

Hydrolysis of Starch and Cellulose • Amylases are extensively applied enzymes which can hydrolyze the glycosidic bonds in starch and related glucose-containing compounds. • There are two major types of amylases- • a-amylase • b-amylase

Glucose Structure

a(1-4) glycosidic linkage between the C1 hydroxyl of one glucose and the C4 hydroxyl of a second glucose The b(1-4) glycosidic linkage is represented as a "zig-zag" line, but one glucose residue is actually flipped over relative to the other

Amylopectin Structure

Hydrolysis of Starch and Cellulose • Starch contains straight-chain glucose polymers called amylose and a branched component known as amylopectin. • The branched structure is relative more soluble than the linear amylose and is also effective in rapidly raising the viscosity of starch solution. • The action of a-amylase reduces the solution viscosity by acting randomly along the glucose chain at a-1,4 glycosidic bonds • a-amylase is often called the starch-liquefying enzyme for this reason.

Hydrolysis of Starch and Cellulose • b-Amylase can attack starch a-1,4 bonds only on the nonreducing ends of the polymer and always produces maltose when a linear chain is hydrolyzed. • Because of the characteristic production of the sugar maltose, b-amylase is also called a saccharifying enzyme. • soluble mixture of starch and b-amylase yields maltose and a remainder of dextrins (starch remnants with 1,6- linkage on the end)

Hydrolysis of Starch and Cellulose • Another saccharifying enzyme, amyloglucosidase(also called glucoamylase) attacks primarily the nonreducinga-1,4 linkages at the ends of starch, glycogen, dextrins, and maltose. (a-1,6 linkages are cleaved by amyloglucosidase at much lower rates) • Sequential treatment with a-amylase and glucoamylase or enzyme mixtures are utilized where pure glucose rather than maltose is desired, e.g., in distilleries and in the manufacture of glucose syrups (corn syrup) and crystalline glucose.

Common Application of Amylase Preparation

Hydrolysis of Starch and Cellulose • The sources of amylases are very numerous. • Amylases are produced by a number of bacteria and molds – • e. g., amylase produced by Clostridium acetobutylicumwhich is clearly involved in the microbial conversion of polysaccharides to butanol and acetone.

Hydrolysis of Starch and Cellulose • Commercial amylase preparations used in human foods are normally obtained from grains, e.g., barley, wheat, rye, oats, maize, sorghum, and rice. • The ratio of saccharifying to liquefying enzyme activity depends • on the particular grain • and upon whether the grain is germinated.

Hydrolysis of Starch and Cellulose • In the production of malt for brewing, the ungerminated seeds are exposed to a favorable temperature and humidity so that rapid germination occurs, with resulting large increase in a-amylase. • The germinated barley is then kiln-dried slowly; • this halts all enzyme activity without irreversible inactivation. • The dried malt preparation is then ground, and its enormous liquefying and saccharifying power is utilized in the subsequent yeast fermentation. • to convert starches to fermentable sugars.

Invertasehydrolyzes sucrose and polysaccharides containing a b-D-fructofuranosyl linkage. • The hydrolyzed sucrose solution containing fructose and glucose rotates a polarized light beam in the direction opposite that of the original solution. • The partially or completely hydrolyzed solution allows two properties desirable in syrup and candy manufacturing: • a slightly sweeter taste than sucrose • and a much higher sugar concentration before hardening.

Hydrolysis of Disaccharides • Maltose1. Maltose + H2O -*--> glucose + glucose * = enzyme; in this case maltaseEnzymes end in -ase • SucroseSucrose + H2O -*-> glucose + fructose * = sucrase • Hydrolysis of LactoseLactose + H2O -*-> galactose + glucose * = lactase

Hydrolysis of Cellulose • For cellulase • Trichodermafungi are commonly used at the present time. • They are thoroughly developed and characterized at present. • There are three major classes of enzymes for different substrates and products • Exo-b-1.4-cellobiohydrolase (CBH) • Endo-b-1.4-glucanase • b-glucosidase

Hydrolysis of Cellulose

Hydrolysis of Cellulose • Many other microorganisms including the molds bacteria produce cellulases with distinctive activities and properties. e.g.- • Fusariumsolani, • Aspergillusniger, • Penicilliumfunicolsum, • Sporotrichumpulverulentum, • Cellulomonasspecies, • Clostridium thermocellum, • and Clostridium thermosaccharolyticum

Cellulose Structure • Cellulose structure e.g., crystallinity, specific surface area and degree of polymerization are important • Cellulose structure can be altered by a variety of pretreatments such as ball or compression milling, g-irradiation, pyrolysis, and acidic or caustic chemicals.

Esterase Applications • Cleave or synthesize ester bonds to yield an acid and an alcohol • Anaerobic waste digestion • Meat processing

Proteolytic Enzymes • Attack nitrogen-carrying compounds, particularly proteins • Dry cleaning • Detergents • Meat processing • Cheesemaking

Lecture 6- Applied Enzyme Catalysis